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JPET #234914
Coproporphyrins in plasma and urine can be appropriate clinical biomarkers to
recapitulate drug-drug interactions mediated by OATP inhibition
Yurong Lai, Sandhya Mandlekar, Hong Shen, Vinay K. Holenarsipur, Robert Langish, Prabhakar
Rajanna, Senthilkumar Murugesan, Nilesh Gaud, Sabariya Selvam, Onkar Date, Yaofeng Cheng,
Petia Shipkova, Jun Dai, William G. Humphreys and Punit Marathe
Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, 3551 Lawrenceville Road,
Princeton, NJ 08540 (Y.L, H.S., R.L., Y.C., P.S., J.D., W.G.H, P.M.); Bristol-Myers Squibb India Pvt. Ltd.,
Biocon BMS R&D Center, Bangalore, India (S.M.); Biocon BMS R&D Center, Syngene International Ltd.,
Bangalore, India(V.H.K., P.R., S.M., N.G., S.S, O.D).
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Running title: Coproporphyrins are clinical biomarkers for OATP inhibition
Corresponding to Dr. Yurong Lai
Bristol-Myers Squibb Company
Route 206 & Province Line Road, Princeton, NJ 08543-4000
E-mail: yurong.lai@bms.com
Tel: 1-609-252-6365
Number of words:
Abstract: 248 (≤250)
Introduction: 747
Discussion: 1302
Number of tables: 2
Number of figures: 4
Number of references: 52
Abbreviations: Cmax, maximum plasma concentration; AUC, area under the concentration-time
curve; CLr, renal clearance; OATP, organic anion transporting polypeptide; CP-I, coproporphyrin
I; CP-III, coproporphyrin III; RIF, rifampicin; RSV: rosuvastatin; LC-MS/MS, liquid
chromatography–tandem mass spectrometry;
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ABSTRACT:
In the present study, an open label, three-treatment, three-period clinical study of rosuvastatin
(RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12
male healthy subjects to determine if CP-I and III could serve as clinical biomarkers for OATP1B1
and 1B3 that belong to solute carrier organic anion gene subfamily (SLCO1B1 and 1B3).
Genotyping of human OATP1B1 gene was performed in all 12 subjects and confirmed absence
of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III
prior to drug administration were 0.91±0.21 and 0.15±0.04 nM, respectively, with minimum
fluctuation over the three periods. CP-I was passively eliminated while CP-III was actively
secreted from urine. Administration of RSV caused no significant changes in the plasma and
urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration
(Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as
compared to the predose values. The area under the plasma concentration curves (AUC0-24hr) of
CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively when compared to
RSV alone. In agreement with this finding, Cmax and AUC0-24hr of RSV increased by 13.2- and 5-
fold, respectively, when RIF was co-administered. Collectively, we conclude that CP-I and CP-III
in plasma and urine can be appropriate endogenous biomarkers specifically and reliably
reflecting OATP inhibition and thus the measurement of these molecules can serve as a useful
tool to assess OATP DDI liabilities in early clinical studies.
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INTRODUCTION
Human hepatic organic anion-transporter polypeptides (OATPs) are expressed on the
basolateral membrane of hepatocytes and responsible for the hepatic uptake of numerous
drugs and endogenous compounds. These transporters are the most important players in
disposition of a wide range of drugs with active hepatic uptake as the rate determining step for
drug clearance (Kalliokoski and Niemi, 2009; Niemi et al., 2011; Yoshida et al., 2012; Shitara et
al., 2013a; Varma et al., 2015). OATP inhibition can cause drug-drug interactions (DDIs) leading
to drug attrition or limitation of use in the clinic for both metabolically stable and unstable
drugs. Understanding the potential pharmacokinetic (PK) interactions of a new chemical entity
with commonly administered co-medications is important for patient safety and is required as
part of the regulatory approval process for drugs unless OATP inhibition can be definitively
ruled out based on preclinical experiments.
Per the current regulatory guidance, the approach for detecting the potential for OATP-
mediated drug interactions for a new chemical entity (NCE) in the clinic is to conduct a drug
interaction study using a probe substrate of OATP, such as rosuvastatin (RSV). In general,
whether to conduct these expensive clinical DDI trials is determined by a static mathematical
approach through computing the ratio of unbound maximum portal vein inhibitor
concentration in vivo for the NCE against the in vitro OATP IC50. While the current approaches in
the regulatory guidance are likely sufficient to minimize the chance of a false negative
interaction, the potential for a high rate of false-positive predictions has been a particular
concern in the pharmaceutical industry (Prueksaritanont et al., 2013; Tweedie et al., 2013). The
estimation is challenged or compromised by the uncertainty of protein binding values and the
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rate and fraction of oral absorption, as well as the mechanisms of transporter inhibition, e.g
long lasting effects (Shitara et al., 2013b). Challenges also remain in our ability to overcome
limitations of in vitro IC50 assessment using different probe substrates, incubation conditions
and variability when using different expression systems (Amundsen et al., 2010; Izumi et al.,
2013; Shitara et al., 2013b; Izumi et al., 2015).
From the industry perspective, the potential PK interactions should be addressed as
early in drug development as possible, allowing important plans and decisions to be made
about compound selection for clinical development prior to significant investment of late-phase
clinical trials. In particular the dose selection remains a dilemma in the design of a clinical DDI
study, when the clinically efficacious dose is yet to be defined. Collectively, in order to avoid
expensive false-positive clinical trials or the risk of late stage failures, sensitive and specific
endogenous biomarkers that can be measured during phase I dose escalation trials would have
substantial benefits for the pharmaceutical industry.
Previously, we conducted preclinical studies to investigate coproporphyrin-I and III (CP-I
and CP-III) in plasma and in urine as markers of OATP activity. CP-I and III are porphyrin
metabolites arising from heme synthesis, and appear to be substrates for human and monkey
OATP1B1 and 1B3 (Bednarczyk and Boiselle, 2015; Shen et al., 2016a). They are stable in the
systemic circulation and in metabolically active tissues e.g hepatocytes (Bednarczyk and
Boiselle, 2015; Shen et al., 2016a), and eliminated in bile and urine as intact forms (Aziz et al.,
1964a; Aziz et al., 1964b; French and Thonger, 1966; Koskelo et al., 1966; Koskelo and
Toivonen, 1966; Koskelo et al., 1967; Aziz and Watson, 1969; Ben-Ezzer et al., 1971; Kaplowitz
et al., 1972). Changes in CPs’ elimination in the urine are found to be related to hepatic
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transporter function. For example, Rotor’s syndrome, a genetic human disease with complete
and simultaneous deficiencies of OATP1B1 and OATP1B3 (van de Steeg et al., 2012), is
diagnosed by a marked preponderance of CP-I over CP-III in the urine (Ben-Ezzer et al., 1971;
Wolkoff et al., 1976). In cynomolgus monkeys, the area under the plasma concentration curves
(AUC) of CP-I and CP-III are markedly increased following the administration of OATP inhibitors,
cyclosporine A or Rifampin (RIF). The results suggest that both CP-I and CP-III in plasma and
urine may be novel clinical endogenous biomarkers for assessing OATP-mediated DDIs.
In the present study, an open label, three treatment, three-period oral comparative
bioavailability study was conducted to elucidate whether CP-I and CP-III in plasma and urine are
sensitive, specific and reliable probes reflecting hepatic OATP inhibition. RSV was administered
in one arm as a sensitive probe for OATP function and RIF was administered in two arms as a
potent inhibitor of OATP function.
MATERIALS AND METHODS
Chemicals and drugs.
Coproporphyrin I dihydrochloride (CP-I, 97%) and coproporphyrin III dihydrochloride (CP-III,
97%) were purchased from Frontier Scientific Inc. (Logan, UT, USA). Isotopically labeled CP-I
sodium bisulfate salt (15
N4-CP-I, 98%), used as an internal standard, was purchased from
Toronto Research Chemicals (North York, Canada). Pooled, stock human plasma (3X charcoal
stripped) was purchased from Bioreclamation VT (Westbury, NY, USA). HPLC grade acetonitrile,
HPLC grade water, ethyl acetate, formic acid and bovine serum albumin (BSA) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Artificial urine was purchased from Pickering
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Laboratories, cat #: 1700-0017 (Mountainview, CA, USA). HPLC grade methanol, formic acid and
acetonitrile were purchased from Sigma Aldrich (St. Louis, MO). HPLC water was obtained from
a Barnstead Nanopure deionizing system (Thermo Scientific, Waltham, MA).
RIF 600 mg capsules (RCIN) were obtained from LUPIN (LUPIN Pharmaceutical, MD). RSV 5
mg tablets (Crestor®) were from AstraZeneca (Bangalore, India). Analytical reference standard
for RIF was purchased from Angene International (London, UK) and for RSV from Apollo
Scientific Limited (Stockport, UK). Control human plasma and human urine for the preparation
of calibration standards (CS) and quality controls (QC) of RIF and RSV were procured from
Syngene International Limited (Bangalore, India). Ritonavir was from USP (Rockville, MD). All
other reagents and solvents were of High performance liquid chromatography (HPLC) grade,
unless specified, and purchased from Sigma-Aldrich Corporation (Bangalore, India). All
compounds were of analytical grade (≥ 95% purity).
Subject Selection:
Healthy male volunteers aged between 18 to 45 years, normal BMI [18.50 to 24.99 kg/m2]
and with minimum weight of 50 kg with no clinically relevant conditions identified from the
medical history, physical examination, electrocardiography or chest X-ray were eligible for
inclusion. Volunteers were excluded if any clinically relevant laboratory abnormality was
identified in clinical chemistry tests (including tests of hepatic and renal biochemistry),
hematology tests, urinalysis or if values for total bilirubin, alanine aminotransferase, aspartate
aminotransferase or alkaline phosphatase were outside the normal reference ranges at the
start of the trial.
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Clinical study design.
This was an open label, three-fixed-treatment, three-period, single dose crossover study in
twelve healthy male Indian adult subjects under fasting condition. The study protocol and the
informed consent were reviewed and approved by the institutional independent ethics
committee of Syndgene International Limited. The study was conducted in accordance with
relevant Syngene clinical development SOPs, ICH 'Guidance on Good Clinical Practice',
Declaration of Helsinki (Fortaleza, Brazil 2013), CDSCO guidelines, ICMR guidelines, and other
applicable regulatory requirements. The clinical part of the study was conducted at one clinical
site (Human Pharmacology Unit, Syngene International limited Clinical development, Tower I,
Biocon Park, Electronics City, Phase-II, Bangalore – 560 100, India). Subjects agreed to refrain
from use of any medicines for 14 days preceding the study. Each subject provided written
informed consent prior to initiation of study procedures.
The trial consisted of three periods separated by a 7 day washout period between two
periods. Volunteers received RIF capsules 600 mg, RSV tablets 5 mg or RIF capsules 600 mg plus
RSV tablets 5 mg in period 1, 2 and 3 (P1, P2 and P3), respectively. In each period,
investigational products were administered orally with 240 mL of water after an overnight fast
of at least 10 hr. Standardized meal was provided 4 hr post-dose. Water was provided ad
libitum except for 1 hr before and 1 hr after dose administration. In each period of the study,
twelve blood samples (~3 mL) were collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and
24 hr post-dose. Samples were collected into tubes containing K2EDTA as anticoagulant and
centrifuged (2600 g at 4 °C for 10 minutes). The plasma was separated into two aliquots. To one
aliquot consisting of 500 μL of plasma, 50 μL of 1 M ammonium acetate buffer (pH 5.0) was
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added and mixed for the measurements of RSV and RIF concentrations in the plasma. The other
aliquot was used for CP measurements. All plasma samples were stored at -70 ± 10°C until
analysis.
At predose, about 120 µL of blood was collected from each subject on FTA elute cards as
dry blood spot and the cards were dried for 2 hr at ambient conditions. These samples were
used for genotyping of SLCO1B1 polymorphism (A388>G and T521>C).
Urine samples were collected in each period during predose (-7 to 0hr), and at about 0 to
7hr and 7 to 24hr post-dose. Multiple urine samples in each interval were pooled, and the total
volume of urine in each interval was recorded. An aliquot of 20 mL urine sample was
transferred into two polypropylene tubes and stored at -70 °C until analysis.
Safety and tolerability were assessed with clinical evaluations, which included a physical
examination and laboratory assessments. Total and direct bilirubin in plasma were measured by
a Beckman Coulter AU 400 automated clinical chemistry analyzer (Brea, CA) according to
manufacturer’s instructions. Indirect bilirubin was calculated by subtracting direct bilirubin
from total bilirubin. Adverse experiences were monitored throughout the study.
Quantification of CPs I and III by LC-MS/MS:
All samples were kept from light exposure as much as possible during sample
preparation. Urine samples were diluted using artificial urine (Pickering Laboratories) with 0.1%
BSA (bovine serum albumin), to ensure that the MS response was within the range of the
calibration curve. Urine or plasma aliquots (100 µL) were transferred to a 1 mL 96-well plate
and then mixed with 50 µL internal standard solution (1.5nM 15
N4-CP I in 12 M formic acid for
plasma samples, 2.5nM 15
N4-CP-I in 6 M formic acid for urine samples) and 500 µL of ethyl
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acetate. After vortex-mixed, the plate was centrifuged at 4000 rpm for 15 mins. The
supernatant (380 µL) was transferred to a 1 mL 96-well plate and dried using a nitrogen plate
dryer with a heat setting of 55 °C. Samples were then reconstituted with 60 µL of 1M formic
acid for LC-MS/MS analysis.
The sample analysis was conducted on a SCIEX 5500 tandem mass spectrometer
(Applied Biosystems/MDS SCIEX, Toronto, Canada), coupled to a CTC Analytics UPLC FLUX pump
and a CTC Analytics HTS PAL autosampler (CTC Analytics, Switzerland). Samples (10 µL) were
injected onto an Ace Excel 2 C18 UPLC, 2.1 x 150mm (particle size 1.7 µm) (Advanced
Chromatography Technologies, Aberdeen, Scotland) and eluted by a gradient program of 20% B
to 60% B in 4 min, 60%B to 100%B in 0.5 min and held 100%B for 0.5 min. The mobile phase
was a mixture of 0.1% formic acid in water (A) and 98% acetonitrile in water containing 0.1%
formic acid (B). The column temperature was maintained at 65°C and the flow rate was 0.5
mL/min. The mass spectrometer was operated in positive, multiple reaction monitoring (MRM)
mode. The MRM precursor/product ion transitions were: m/z 655.3 > 596.3 for CP-I and III, and
m/z 659.3> 600.3 for the internal standard, 15
N4-CP-I. The instrument settings on the mass
spectrometer were as follows: declustering potential (DP) 130 V; collision energy (CE) 65 V;
dwell time 25 ms. All peak integration and data processing were performed using SCIEX Analyst
1.6.2 (Applied Biosystems/MDS SCIEX, Toronto, Canada). The LC-MS/MS method validation and
quality controls are presented in supplementary material.
Quantification of RSV and RIF by LC-MS/MS.
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Sample extraction for RSV and RIF was conducted in 96-well plates using protein
precipitation with acetonitrile. In brief, plasma and urine samples (30 µL) for RSV measurement
were mixed with 125 µL ice-cold acetonitrile containing 200 nM of ritonavir as an internal
standard in a hydrophilic solvinert plate (Millipore Corporation, Billerica, USA). Similarly, an
aliquot of 20 µL plasma and urine samples (50X diluted in control plasma) for RIF measurement
were mixed with 180 µL ice-cold acetonitrile containing 200 nM of ritonavir in a hydrophilic
solvinert plate. Samples were vortexed and centrifuged at 4°C, 4000 rpm for 5 minutes. The
supernatant (5 µL for RSV measurement and 3 µL for RIF measurement) was injected on to LC-
MS/MS.
The liquid chromatography system, ACQUITY UPLCTM
consisted of ACQUITY binary
solvent manger and ACQUITY sample manger with sample organizer (Waters Corporation,
Milford, USA). Chromatographic separation was achieved by gradient elution on an Acquity C18
BEH, 1.7µm, 2.1*50mm column (Waters Corporation, Milford, USA) maintained at 40°C. The
mobile phase was a mixture of 0.1% formic acid in 10 mM ammonium formate (A) and 0.1%
formic acid in acetonitrile (B). The gradient for RSV was set as follows: 10% B to 95% B in 2 min,
held 95%B for 0.4 min, 95% B to 10% B in 0.1 min and held 10%B for 0.5 min. The flow rate was
0.6 mL/min. The gradient for RIF was set as follows: 2% B to 50% B in 0.5 min, 50% B to 100% B
in 0.01 min, held 100%B for 0.09 min, 100% B to 2% B in 0.05 min and held 2%B for 0.35 min.
The flow rate was 0.8 mL/min.
Mass spectrometric detection for RSV and RIF was performed on an AB Sciex 5500
QTRAP and 4000 QTRAP (Applied Biosystems, Foster City, USA), respectively, equipped with
electrospray ionization source. The mass spectrometer was operated in positive ion mode and
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MRM transitions were used for RSV (m/z 482.1>258.07) and RIF (m/z 823.4>791.3) detection.
The instrument settings on the mass spectrometers were as follows: ion spray voltage: 5.5 kV;
temperature: 550 °C; declustering potential (DP): 100 V; collision energy (CE) 47V and 25V (for
RSV and RIF, respectively), entrance potential (EP) 10V, and collision cell exit potential (CXP):
15V. Peak integration and data processing were performed using AnalystTM
version 1.6.2.
(Applied Biosystems, Foster City, USA). The LC-MS/MS method validation and quality controls
are presented in supplementary material.
Identification of variants in OATP1B1 gene
Blood was spotted from each subject on FTA Elute Micro Cards (WB120401 from GE Life
Sciences) and dried. One 6 mm sample from each spot was punched out and taken in a 1.5ml
sterile centrifuge tube to which 500 µL of nuclease free water was added and vortexed for
about 5 seconds to rinse the punch. Water was removed using a sterile pipette followed by
addition of 100 µL nuclease free water and heating at 95oC for 30 minutes. The samples were
removed from the heat block and vortexed for 30 seconds. The samples were centrifuged for
30 seconds to separate the matrix and eluate containing purified DNA. The eluate was then
transferred into a fresh tube.
SNP probe sets (rs2306283 for Asn130Asp and rs4149056 for Val174ala for OATP1B1
gene) were procured from Thermo Fischer Scientific (Grand Island, NY). PPIA probe mix
(NM_021130.3) was used as positive control for each sample. Taqman® assay was performed
using 5 µL of iQTM
multiplex powermix (Cat #172-5849, Biorad, Hercules, CA), 1 µL of DNA
eluate from each sample, 0.5 µL of probe mix and 3.5 µL of nuclease free water. PCR reactions
were conducted in triplicate for each probe set in a 384-well hard shell plate using CFX 384 Real
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time PCR detection system from Biorad. Reaction conditions were as follows: enzyme
activation at 95oC for 3 minutes, denaturation at 95
oC for 15 seconds and annealing/extension
at 60 o
C for 1 min and for a total of 50 cycles. The probes contained a fluorescent reporter dye
(VIC specific for allele”1”, and FAMTM
specific for allele “2”) attached to its 5’ end and a
quencher dye at its 3’ end. Allelic discrimination was analyzed based on relative fluorescence
from the probe sets using CFX manager software from Biorad. The presence of a VIC only
fluorescent signal represents homozygosity for allele “1” e.g 388A and a FAM only fluorescent
signal indicates homozygosity for allele “2” e.g 388G. The presence of both signals suggests
heterozygosity of allele 1 and 2.
Pharmacokinetic Analysis.
The PK parameters were obtained by noncompartmental analysis of plasma
concentration vs. time data (KINETICA™ software, Version 4.4.1, Thermo Fisher Scientific
Corporation, Philadelphia, PA). The maximum plasma concentration (Cmax) and time for Cmax
(Tmax) were recorded directly from experimental observations. The area under the plasma
concentration time curve (AUC) was calculated using the mixed log-linear trapezoidal rule up to
the last quantifiable concentration (Clast). Estimations of AUC and terminal elimination rate
constant were made using a minimum of three time points with quantifiable concentrations.
Concentrations below the limit of quantitation were considered as zero for calculations.
Renal clearance (CLr) was estimated by the following equations:
tAUCo
XCL
−= t-0
R
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where X0-t is the cumulative amount excreted in the urine during the time interval (0~t).
Statistical Analysis.
To test for statistically significant differences among multiple days for CP concentrations
in the plasma and urine excretion, one-way ANOVA followed by Dunnett comparisons was
performed. Student’s t test was used to assess the statistical significance of differences
between 2 sets of data. All statistical analyses were performed using Prism version 5.0
(GraphPad Software, Inc.; San Diego, CA). A p-value of less than 0.05 was considered to be
statistically significant.
RESULTS
Study enrolment:
Single doses of RSV administered either alone or co-administered with RIF were well
tolerated in healthy subjects. No adverse events, serious adverse experiences, laboratory
adverse experiences or events of clinical interest were reported during the entire duration of
the study. No subjects were discontinued by the study investigator. No clinically significant
changes in the measured values of blood pressure, pulse rate, respiratory rate and oral
temperature were observed during the entire duration of the study which could be related to
the study drug. Twelve subjects were enrolled at the beginning of the study. Subject 7 could not
attend period 2, but he visited the facility and completed period 3. Subject 8 completed period
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1 and period 2 but dropped out of the study prior to period 3 due to personal reasons. All other
subjects completed the entire study. Genotyping of two common single nucleotide
polymorphisms (SNP) of human OATP1B1 gene, A388G (Asn130Asp), and T521A (Val174Ala)
was performed in all 12 subjects. OATP1B1*5 and OATP1B1*15 mutations were absent in all 12
subjects. Nine subjects were heterozygous and three subjects were homozygous for
OATP1B1*1b.
Pharmacokinetics of RIF
The mean plasma concentration vs. time profiles of RIF following a single oral dose of
600 mg RIF were similar to those observed in combination with oral administration of 5 mg RSV
(Figure 1 and Table 1). RIF achieved a maximum plasma concentration (Cmax) of 26.7 and 30.6
µM at 2.5- and 2.2 hr post dose in period 1 and period 3, respectively. The average plasma
concentrations of RIF from the combination treatment were about 6.8 µM and 0.7 µM at 12
and 24 hr post-dose, respectively. At 12 hr post dose, RIF free concentration (89% protein
bound) was 0.75 µM, which is above the IC50s of human OATP1B1 (0.55±0.07 µM) or OATP1B3
(0.46 ±0.13 µM) (Shen et al., 2013).
Pharmacokinetics of RSV
The mean plasma concentration vs. time profiles and pharmacokinetic parameters of
RSV following a single oral dose of 5 mg RSV (period 2) or in combination with 600 mg RIF
(period 3) are depicted in Figure 2 and Table 1. The Cmax and AUC0~24h of RSV following RSV
dose alone were 9.17±3.85 nM and 75.6 ± 26.5 nM*h, respectively. RIF markedly increased the
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Cmax and AUC0~24h of RSV by 13.2- and 5-fold, respectively. Time to maximal concentration (Tmax)
and terminal half-life (T1/2) of RSV decreased significantly by the co-administration of RIF (Table
1). As expected, urinary excretion of RSV was minimum accounting for about 5% of total dose.
RIF increased the amount of RSV in urine to 24 % of total dose but had no impact on CLr of RSV
(Table 1).
CP-I and CP-III concentrations in plasma and urine
Mean plasma concentration vs. time profiles of CP-I and CP-III after RIF alone (period 1),
RSV alone (period 2) or in combination (period 3) are shown in Figure 3. Both CP-I and CP-III
concentrations in plasma were not affected by RSV, as compared to the predose values (Table
2). Changes in CP-I and CP-III plasma concentrations in period 1 are considered to be
independent of time (statistically not significant); therefore, Cmax and Tmax were not computed
for CP-I and CP-III for this treatment (Table 2). There was no significant difference in the
predose plasma concentrations and urine excretion (Xe(-7~0 hr)) of CP-I and CP-III in the three
periods (Table 2), suggesting that CP-I and CP-III in the plasma and urine at baseline are
constant. The ratio of CP-I vs. CP-III at baseline was about 6:1 in the plasma, and about 1:1 in
the urine.
Following administration of a single dose 600 mg RIF alone or in combination with 5 mg
RSV, a significant increase in CP-I Cmax (5.7~5.9-fold) was observed when compared to the
predose concentration. Likewise, RIF, either alone or in combination with RSV, significantly
increased CP-III Cmax by a 5.4~6.5-fold. Plasma AUC0~24 of CP-I and CP-III were also significantly
increased by 4- and 3.4-fold, respectively, following RIF (Figure 3 and Table 2), compared to RSV
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alone. Plasma concentrations of both CP-I and CP-III returned to predose concentrations after
24 hr post RIF dose, the time at which free plasma concentration of RIF is lower than the OATP
IC50s (Shen et al., 2013).
The cumulative amount of CP-I excreted in urine Xe (0–24 h) increased by 3.6-fold (RIF
alone) or 3.4-fold (RIF+RSV), whereas CP-III urinary amount increased by 1.6-fold (RIF alone) or
1.4-fold (RIF+RSV), respectively (Table 2). As expected, the renal clearance of CP-I was not
altered by RIF (Table 2). The administration of RIF alone or in combination with RSV reduced the
CLr of CP-III; however, the reduction was not statistically different due to the large inter-
individual variation (Table2).
Total, direct and indirect bilirubin in human plasma
As indirect bilirubin is a substrate of OATP transporters and increased plasma bilirubin in
cynomolgus monkeys and rats after RIF administration has been reported (Chu et al., 2015), we
further examined total, direct and indirect bilirubin in plasma of human subjects receiving RSV
alone (period 1) or the combination of RIF and RSV (period 3). As shown in Figure 5, a slight
(~32%) but statistically significant increase in total bilirubin was detected in the combination
treatment, in comparison with RSV alone. The increase in total bilirubin appeared to be
contributed by indirect bilirubin, which was increased by about two-fold following the
combination treatment. In contrast, the changes in direct bilirubin in plasma were similar in the
two treatments. No abnormal changes in other liver enzymes e.g. alanine miotransferase (ALT)
were reported in any subject.
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DISCUSSION
CP-I and CP-III are actively taken up by human hepatocytes and human embryonic
kidney 293 cells overexpressing human OATP1B1 or 1B3 protein (Bednarczyk and Boiselle,
2015; Shen et al., 2016a). The results suggested that CPs could be potential endogenous
biomarkers of OATP activity in vivo (Benz-de Bretagne et al., 2011; van de Steeg et al., 2012).
Indeed, the plasma concentrations of both CP-I and CP-III in cynomolgus monkeys were
markedly increased following administration of OATP inhibitors, cyclosporine A (100 mg/kg
oral) or RIF (15 mg/kg oral) (Shen et al., 2015; Shen et al., 2016b). The observations in monkey
could explain the clinical findings that CsA- or RIF -induced porphyria in patients is likely due to
the inhibition of OATP transporters (Millar, 1980; Hivnor et al., 2003). In addition, changes in
plasma CP-I and CP-III in cynomolgus monkey are in line with RSV exposure increase after co-
administering with OATP inhibitors, suggesting CPs could be biomarkers for in vivo OATP-
mediated DDIs (Shen et al., 2016a).
In the current clinical study, we found that plasma concentrations of both CP-I and III
were relatively stable at predose and after administration of RSV, and increased following
administration of RIF. In line with the increase in RSV exposure in human subjects receiving a
single oral dose 600 mg RIF, Cmax of CP-I and CP-III increased by 5.7~5.9- and 5.4~6.5 fold, while
AUC0-24hr increased by 4 and 3.4-fold, respectively. Although the Cmax increase of CP-I and III was
less than that of RSV (5.4~6.5 fold vs. 13.2 fold), the changes in AUC are comparable to AUC
changes of RSV found in the current study (Table 1) and another report (4.4-fold)
(Prueksaritanont et al., 2014). RSV is specifically distributed in the liver, where it is eliminated
into the bile by cannalicular efflux transporters (Nezasa et al., 2002). The inhibition of OATP
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function could decrease both systemic clearance and volume distribution of RSV. Indeed, CL/F
of RSV was decreased by 4.7-fold following the administration of RIF, while Vss/F decreased by
8.7-fold resulting in the decrease of T1/2 (0.7-fold). As AUC is independent of Vss, greater
changes in the Cmax (13.2-fold) over AUC0~24hr (5-fold) of RSV can be explained by the decrease
in Vss.
Dubin-Johnson Syndrome, an autosomal recessive genetic disorder leading to deficiency
of human multidrug resistance-associated protein 2 (MRP2), causes an increase of ratio of
urinary CP-I to CP-III. Urinary elimination of CPs is dependent on ABCC2 polymorphisms and
represents a potential biomarker of MRP2 activity in humans, suggesting that CPs is
substrates for MRP2. Furthermore, in vitro data show that rifampicin is substrates for P-
glycoprotein, MRP2 and Breast cancer resistance protein (BCRP)
(https://www.druginteractioninfo.org). Although clinical DDI results of pitavastatin suggested
that inhibition of OATP1B-mediated hepatic uptake of pitavastatin is primarily responsible for
the increased pitavastatin exposure in the presence of RIF, and the contribution of inhibition for
MRP2 to the rifampicin-pitavastatin interaction is considered small (Prueksaritanont et al.,
2014), the potential impact of MRP2 inhibition on plasma CPs cannot be completely excluded
and further investigations are needed.
At predose, CP-I and CP-III amounts were about the same in human urine and the ratio
of CP-I over total CPs close to previously reported (0.29~0.44) (Koskelo et al., 1966; Koskelo et
al., 1967; Gebril et al., 1990), but different from the profiles in monkey where CP-III was much
higher than CP-I (Shen et al., 2016a). Urinary excretion of CP-I increased (3.6 fold), to a similar
extent as that of CP-I AUC0~24hr (4.0-fold) after RIF treatment, as compared to RSV alone. The
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increase in urinary excretion of CP- III (1.6-fold), was less compared to CP-I after RIF (3.3-fold),
resulting in the preponderance of CP-I over CP-III in urine. The result is consistent with the
abnormal distribution of CP isomers reported in the urine of patients with Rotor syndrome
(Ben-Ezzer et al., 1971; Wolkoff et al., 1976). RIF treatment appeared to decrease CLr of CP-III,
suggesting involvement of renal transporters. (Koskelo et al., 1966; Koskelo et al., 1967; Gebril
et al., 1990)Although CP-III, but not CP-I appeared to be transported by organic anion
transporter (OAT) 1 (Bednarczyk and Boiselle, 2015), further characterization of transporter(s)
responsible for active renal secretion of CP-III and its association with genetic polymorphism is
necessary. Overall, since CP-I is passively cleared in the urine, and the urinary output is
proportional to the AUC increase, CP-I in the urine may have better value than CP-III, as an in
vivo biomarker for OATP function without complication by renal transporters.
Two SLCO1B1 SNPs OATP1B1*5 and *15 could cause decreased cell surface expression
and/or affect in vitro and in vivo transport activities, and are associated with increased plasma
exposure (AUC) of substrates. (Nishizato et al., 2003; Mwinyi et al., 2004; Pasanen et al., 2008;
Seithel et al., 2008). For example, plasma exposure of repaglinide or statins (AUC) is 1.5~3 fold
higher in heterozygous or homozygous subjects for SLCO1B1 521CC genotype (Val174Ala)
compared with subjects with SLCO1B1 521TT genotype (Kivisto and Niemi, 2007; Kalliokoski et
al., 2008; Niemi et al., 2011). OATP1B1*1b haplotype is reported to associate with increased
activity of OATP1B1 (Katz et al., 2006) and lower AUC of pitavastatin in Japanese subjects
(Maeda et al., 2006); controversially the observed pharmacokinetic differences for RSV
between Asians and Caucasian subjects is not due to SLCO1B1*1b genotype (Lee et al., 2005;
Choi et al., 2008). To our knowledge, there is no data describing the association between
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SLCO1B1 SNPs and plasma concentration of CP-I and CP-III. In the present study, three subjects
were found to be OATP1B1*1b homozygous, and nine OATP1B1*1b heterozygous. Although no
significant differences in CP-I and CP-III baseline between homozygous and heterozygous
OATP1B1*1b were found (the Table 1 and Figure 1 presented in supplementary material), the
small study size did not allow to draw any conclusions of the impact of OATP1B1 genotypes on
the plasma CPs. Further investigation of impact of OATP genetic polymorphisms on CP plasma
concentration is warranted.
OATP1B1 and 1B3 have been shown to transport both unconjugated and conjugated
bilirubin in vitro (Kalliokoski and Niemi, 2009). OATP inhibition can cause hyperbilirubinemia
(Campbell et al., 2004). Indeed, bilirubins are markedly increased in the plasma in cynomolgus
monkeys and rats after administration of RIF (Chu et al., 2015; Watanabe et al., 2015); thus, the
authors proposed bilirubin as a biomarker for hepatic OATP inhibition. However, in addition to
the biochemical defect leading to reduced hepatic uptake of conjugated and unconjugated
bilirubin, other factors such as impaired efflux transporters (MRP2 and MRP3) and enzyme
activity (UGT1A1) may also result in changes in bilirubin plasma concentration. Furthermore,
serum bilirubin, along with bile acids and other liver enzymes, are altered with drug induced
liver injuries (Ozer et al., 2008). In the present study, we showed that the increase in total and
indirect bilirubin was marginal (<2-fold). The increase in total bilirubin could be attributed to
the increase in indirect bilirubin in plasma suggesting the inhibitory effect occurs on the
sinusoidal side of hepatocytes (OATP uptake), rather than cannalicular efflux (MRP2).
Collectively, the results presented herein showed that there is strong mechanistic
relationship between OATP inhibition and CP-I and CP-III plasma and urine concentrations. As
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aforementioned, although impact of age, gender, contributions of inhibition for hepatic efflux
transporters and genetic polymorphisms etc on CP-I and CP-III remains to be further resolved
by incoming clinical data, CP-I and CP-III in plasma, and CP-I in urine could be surrogate
endpoints for DDIs mediated by OATP inhibition in human. Importantly, CP-I and CP-III
measurements can be incorporated in Phase I dose escalation studies to provide evidence of
OATP inhibition across all doses. We propose that CP-I and CP-III measurements should be
incorporated in the decision tree for OATP DDI risk assessment and thereby inform planning
and design of downstream clinical studies. Further investigations are needed to understand
influence of other intrinsic and extrinsic factors such as age, gender, genetic polymorphism and
organ impairment on CP-I and CP-III plasma and urine concentrations.
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Acknowledgments
The authors wish to thank Dr. Kurex Sidik, for help with statistical analysis; Dr. Anil K., Dr.
Siddangouda Patil and Jaya Patel, Syngene Clinical Development, Kamala Venkatesh and Uday
Kanni, BBRC for bilirubin analysis; and Dr. Hemant Bhutani and Shishir Prasad, BBRC for assay of
formulations; and Dr. Randy C. Dockens for reviewing the protocol of clinical studies. We also
acknowledge Anne Rose, Tongtong Liu for handling clinical samples, and Anthony Marino for
reviewing the manuscript.
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Authorship Contributions
Participated in research design Mandlekar, Shen, Cheng, Shipkova, Humphreys, Marathe and
Lai
Conducted experiments: Langish, Dai, Holenarsipur, Rajanna, Murugesan, Gaud, Selvam, Date
Contributed new reagents or analytic tools: Langish, Dai, Shipkova, Rajanna, Murugesan, Gaud,
Selvam, Date
Performed data analysis: Mandlekar, Shen, Cheng, Holenarsipur, Gaud, Humphreys, Marathe
and Lai
Wrote or contributed to the writing of the manuscript: Mandlekar, Shen, Cheng, Zhang,
Holenarsipur, Rajanna, Dockens, Shipkova, Humphreys, Marathe and Lai
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Figure Legends
Figure 1. Plasma concentration-time profiles of Rifampicin (RIF). Plasma concentrations of RIF
were determined following administration of 600 mg RIF alone (period 1) or a combination of
600 mg RIF and 5 mg RSV (period 3). The data are expressed as mean ± SD of 12 (P1) or 11
(period 3) subjects.
Figure 2. Effects of rifampicin (RIF) on rosuvastatin (RSV) exposure. RSV plasma concentration-
time profiles were determined following oral administration of 5 mg RSV alone (period 2) or a
combination of 5 mg RSV with 600 mg RIF (period 3). The data are expressed as mean ± SD of
11 subjects.
Figure 3. Effects of rifampicin (RIF) on plasma coproporphyrin (CP) concentrations. CP plasma
concentrations were determined following oral administration of 600 RIF alone (period 1), 5 mg
RSV alone (period 2) and the combination of 600 mg RIF and 5 mg RSV (period 3). A, CP-I; B, CP-
III. The data are expressed as mean ± SD of 12 (period 1) or 11 (period 2 and period 3) subjects.
Figure 4. Effects of rifampicin (RIF) on plasma bilirubin. Mean plasma concentrations of total
(A), direct (B), and indirect bilirubin (C) were determined following RSV alone and RSV+RIF
administration. The AUC0~20hr of total, direct and indirect bilirubin are listed in the table. Data
are expressed as the mean ± SD of 11 subjects. *, P<0.05 statistically significant difference
between the subjects administered with RIF alone (period 2) and RIF+RSV (period 3)
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Table 1. Pharmacokinetics parameters of RIF and RSV
Analyte Parameters RIF or RSV alone
(Mean ± SD, n=11)
RSV + RIF
(Mean ± SD, n=11) Ratio
RIF
Cmax (µM) 26.7 ± 10.7 30.6 ± 7.8 0.87
Tmax (h) 2.5 ± 1.7 2.2 ± 0.9 1.2
AUC0-24h (µM*h) 207 ± 70.9 220 ± 57.9 0.94
AUC0-inf (µM*h) 215 ± 79.7 224 ± 61.5 0.95
T1/2 (h) 4.1 ± 1.5 3.5 ± 1.2 1.2
C12h (µM) 9.2 ± 3.5 6.8 ± 3.9 1.35
C24h (µM) 1.0 ± 1.1 0.7 ± 0.8 1.39
RSV
Cmax (nM) 9.2 ± 3.8 106 ± 43.7* 13.2*
Tmax (h) 3.3 ± 1.6 1.8 ± 0.6* 0.7*
AUC0-24h (nM*h) 75.6 ± 26.5 372 ± 119* 5.0*
AUC0-inf (nM*h) 80.3 ± 28.2 375 ± 120* 4.7*
AUC0-12 (nM*h) 62.6 ± 22.8 357 ± 116* 5.8*
T1/2 (h) 5.8 ± 1.3 3.8 ± 0.6* 0.7*
CL/F (mL/min) 2372.4±710.9 506.8±163.7* 4.7
Vss/F (L) 1156.7±567.7 132.3±54* 8.7
CLr (mL/min) 124±52.6 124±42.2 1
Urinary excretion (% of
total dose) 5.2% 24%
Cmax, maximum plasma concentration; C12 and C24, plasma concentration at 12-hr and 24-hr
post-dose, respectively; AUC, area under concentration-time curve. T1/2, terminal half-life.
CL/F, plasma clearance over bioavailability; V/F, volume distribution over bioavailability; CLr,
renal clearance; *, P<0.05 as compared to RSV alone
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Table 2. Comparison of pharmacokinetic parameters of CP-I and CP-III following oral
administration of 600 mg RIF alone, 5 mg RSV alone, or the combination of 5 mg RSV and 600
mg RIF
Parameters
CP-I CP-III
RIF RSV RIF+RSV RIF RSV RIF+RSV
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Cpredose (nM) 0.97±0.24 0.85±0.19 0.91±0.20 0.17±0.04 0.15±0.05 0.14±0.04
Cmax (nM) 5.54 ± 0.80* 0.86 ± 0.181 5.39 ± 0.78* 0.92 ± 0.23* 0.28 ± 0.14* 0.91 ± 0.18*
Tmax (h) 6.00 ± 2.56 N/A 7.36 ± 2.20 5.42 ± 2.35 N/A 4.73 ± 2.20
AUC0-24h (nM*h) 84.2 ± 18.7# 20.9 ± 4.95 84.0 ± 15.7
# 12.6 ± 3.84 3.77 ± 1.20 12.5 ± 2.86
Xe (-7 to 0h) (nmol) 14 ± 5.7 9.7 ± 5.1 11.7 ± 3.6 12.4 ± 7.7 10.5 ± 10.8 13.9 ± 8.4
Xe (0 to 7h) (nmol) 51 ± 10.3 12 ± 4.1 31 ± 13.6 18.7 ± 8.9 8.7 ± 6.7 11.7 ± 6.8
Xe (7 to 24h) (nmol) 91 ± 17.2 27 ± 13.8 106 ± 27.7 45 ± 22.9 30 ± 22.4 41 ± 17.1
Xe (0-24h) (nmol) 142 ± 18.0# 39.0 ± 15.0 136.0 ±32.0
# 63.0 ±30.0 38 ± 28 52 ±21
CLr (mL/min) 29.2 ± 6.77 30.9 ± 10.9 27.5 ± 5.67 87.7 ± 46.2 193 ± 171 73.6 ± 38.3
N of subjects 12 11 11 12 11 11
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Data are expressed as mean ± SD. Cmax, maximum plasma concentration; AUC, area under
concentration-time curve; Xe (-7 to 0h), amount excreted in urine during -7 hr to predose. Xe (0 to
7h) amount excreted in urine during 0 to 7 hr. Xe (7 to 24h), amount excreted in urine during 7 to
24 hr. Xe(0~24hr), amount excreted in urine during 24 hr; CLr, renal clearance; *p < 0.05,
statistically significant difference compared to predose; #, P<0.05, statistically significant
difference compared to RSV alone group. 1,
value represents average concentrations from all
subjects, as the changes of CP-I and CP-III following administration of a single dose RSV are
independent of time.
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